Plasmacytoid dendritic cells (pDC) are the most potent type I interferon (IFN)- producing cells in the body and are considered to be the professional IFN producing cells. pDC become both numerically and functionally deficient in HIV-infected individuals, and this dysfunction is strongly correlated with progression to opportunistic infections. In addition to evidence for important protective roles of the pDC in HIV infection, an overzealous pDC IFN-a response has also been associated with immune activation and HIV pathogenesis. The experiments in this application are based on our observations that peripheral blood pDC in viremic HIV-infected-individuals are not only numerically depleted, but are also functionally altered. Our studies since the last competing submission of this grant indicate that this numerical and functional dysfunction is contributed to by several factors including apoptosis of some circulating pDC, activation and potentially recruitment out of the periphery, as well as replacement of peripheral pDC with newly emigrated cells from the bone marrow. In addition, we have discovered that pDC preferentially take-up pieces of membrane and cytoplasm from live, virus-infected cells vs. uninfected cells and subsequently produce IFN-a and mature in response to this nibbling. Upon interaction with HIV-infected cells, however, this process is subverted, and pDC fuse with the HIV-infected cells. We propose three specific aims to follow-up on these observations: In the first specific aim, we will investigate the requirements for, and consequences of, pDC fusion with HIV-infected cells. We will determine how different viral strains affect IFN-a production and pDC:HIV-infected cell interactions, both as cell-free viruses and as virus-infected cells. Included in these viruses will be early transmitted viruses as well as chronic-stage viruses to be obtained from CHAVI. Imaging and traditional flow cytometry will be central to our experimental approaches. In the second aim, we will determine whether pDC:T cell fusion occurs in vivo and in lymphoid tissues using three distinct approaches: first we will determine whether there is evidence of pDC:T cell fusion in peripheral blood of HIV-infected patients; second, we will determine whether pDC:HIV-infected T cell fusion occurs in tonsil suspension cells and tonsil organ culture; third, in collaboration with Dr. Christel Uittenbogaart of UCLA, we will determine whether pDC:T cell fusion occurs in HIV-infected HIS mice. In the final specific aim, we will investigate the functional significance of the different phenotypic subpopulations of pDC in individuals with and without HIV infection and will investigate pDC in the bone marrow of HIV-infected individuals. Together, the studies proposed in this application are anticipated to provide valuable new information on the biology of human pDC and the mechanisms underlying their dysfunction in HIV-1 infection.